This invention relates to a process for producing a composition containing insolubilized enzyme and/or insolubilized bacterial cells which can be continuously employed for a long period or which can be repeatedly employed many times in a batch system.
Recently the enzyme industry has become important and has made remarkable progress in producing medicine and food by utilizing enzyme or cells to carry out a variety of reactions.
In the prior art, enzyme reaction was effected by using enzyme solution. In this case, however, after the reaction is completed, the enzyme solution employed in the reaction cannot be reused, because the used enzyme solution contains the resulting reaction product. Therefore, since the enzyme solution employed in the one reaction must be removed from the reaction system, a batch system must be used for the enzyme reaction. In other words, in enzyme reactions using enzyme solution, the maximum effectiveness of the enzyme is not obtained.
U.S. Pat. No. 3,860,490 by Guttag discloses the microorganisms are entrapped in a hydrophilic catalyst. However, the Guttag invention relates to the quick or controlled release of living microorganisms such as bacteria, molds, yeast and viruses or to providing limited contact between the microorganism and an environment on which it acts. The object of the Guttag invention is to store living microorganisms so that they can be released or can act in an appropriate area and/or at an appropriate time. That is, the object of Guttag invention is to keep the microorganisms dry or out of contact with air until use.
U.S. Pat. No. 3,859,169 by O'Driscoll et al discloses that enzymes are provided in gels.
U.S. Pat. No. 3,871,964 by Huper et al discloses that polypeptide such as enzymes are rendered water-soluble by bonding to a cross-linked copolymer.
U.S. Ser. No. 606,209 filed on Aug. 20, 1975 by Kaetsu et al, now U.S. Pat. No. 4,194,066, discloses a process for producing polymer-enzyme composition containing insolubilized enzyme and/or bacterial cells, characterized by irradiating the mixture of a vitrificable monomer and enzyme and/or cells by means of an ionizing radiation at a temperature of less than 0.degree. C. The monomers employed in U.S. Ser. No. 606,209 differ from the monomers employed in the present invention. U.S. Ser. No. 688,081 filed on May 19, 1976 by Kaetsu et al, now U.S. Pat. No. 4,193,845, discloses a similar process. The monomers employed in U.S. Ser. No. 688,081 are water-soluble. The ratio of the activity of the enzyme-polymer composition of U.S. Ser. Nos. 606,209 and 688,081 to the activity of an aqueous solution containing the same enzyme (sometimes referred to as the degree of activity maintained) is as low as 60% or less. In addition, when the enzyme-polymer composition prepared according to U.S. Ser. Nos. 606,209 and 688,081 is used for a long period under severe conditions, the enzyme is likely to be released. Therefore, the enzyme reaction can not continuously be carried out for a long period in a column by using said enzyme-polymer composition.
U.S. Pat. No. 3,962,038 to Kawashima et al discloses that enzymes are entrapped in polymers of acrylamide, bisacrylamide, acrylic acid, sodium acrylate, potassium acrylate and calcium acrylate. The monomers employed in Kawashima et al are in a crystalline state at a temperature less than 0.degree. C. Polymerizability of the crystallizable monomer is lowered at a temperature less than 0.degree. C., because molecular motion of the monomer is restricted by formation of the crystal lattice. When a large total dose of ionizing radiation is used for polymerizing the monomer, there is possiblity of deactivation of the enzyme and the bacterial cells. When the monomer is crystallized at a low temperature, the enzyme and cells can hardly penetrate into the monomer phase. Therefore, enzyme or cells are not firmly entrapped in polymer according to the invention of U.S. Pat. No. 3,962,038.
Japanese Patent Publication (laid open) No. 92446/1973 corresponding to U.S. Ser. No. 225,489 filed on Feb. 11, 1972, now abandoned, discloses an article composed of a water-insoluble, hydrophilic polymer in which an enzyme is entrapped, said polymer being polymers obtained from the monomer selected from the group consisting of hydroxy loweralkyl acrylate, hydro loweralkyl methacrylate, hydroxyloweralkoxy loweralkyl acrylate, hydroloweralkoxy loweralkyl methacrylate, vinylpyrrolidone, acrylamide, methacrylamide, N-loweralkylacrylamide, N-loweralkyl methacrylamide, N-hydroxyloweralkyl acrylamide and N-hydroloweralkyl methacrylamide. However, these monomers disclosed in the above Japanese Publication are water-soluble.
In other words, the prior process for entrapping enzyme or bacterial cells had the disadvantages that the enzyme is likely to release from the polymer. Therefore, compositions containing insolubilized enzyme prepared according to the prior processes can be used only for a short period in a continuous process, and can not be repeatedly used many times in a batch process.
A process for insolubilizing enzyme which comprises mixing enzyme with an water-soluble monomer, and then polymerizing the monomer has been proposed. However, enzyme is unstable to heat, so the polymerization must be carried out at a low temperature. But, the polymerizing reactivity of the monomer is lowered at a low temperature.